Rates of sulfate reduction and thiosulfate consumption in a marine microbial mat

Abstract The sulfur cycle in a microbial mat was studied by determining viable counts of sulfate-reducing bacteria, chemolithoautotrophic sulfur bacteria and anoxygenic phototrophic bacteria. All three functional groups of sulfur bacteria revealed a maximum population density in the uppermost 5 mm of the mat: 1.1 × 10⁸ cells of sulfate reducers cm⁻³ sediment, 2.0 × 10⁹ cells of chemolithoautotrophs cm⁻³ sediment, and 4.0 × 10⁷ cells of anoxygenic phototrophs cm⁻³ sediment. Bacterial dynamics were studied by sulfate reduction rate measurements, both under anoxic conditions (dark incubation) and oxic conditions (incubation in the light), and determination of the vertical distribution of the potential rate of thiosulfate consumption under oxic conditions. Sulfate reduction rates in the top 5 mm of the sediment were 566 nmol cm⁻³ d⁻¹ in the absence of oxygen, and 123 nmol cm⁻³ d⁻¹ in the presence of oxygen. In the latter case, the maximum rate was found in the 5–10-mm depth horizon (361 nmol cm⁻³ d⁻¹). Biological consumption of amended thiosulfate was rapid and decreased with depth, while in the presence of molybdate, thiosulfate consumption decreased to 10–30% of the original rate.     

Thermophilic anaerobic oxidation of methane by marine microbial consortia

The anaerobic oxidation of methane (AOM) with sulfate controls the emission of the greenhouse gas methane from the ocean floor. AOM is performed by microbial consortia of archaea (ANME) associated with partners related to sulfate-reducing bacteria. In vitro enrichments of AOM were so far only successful at temperatures ⩽25 °C; however, energy gain for growth by AOM with sulfate is in principle also possible at higher temperatures. Sequences of 16S rRNA genes and core lipids characteristic for ANME as well as hints of in situ AOM activity were indeed reported for geothermally heated marine environments, yet no direct evidence for thermophilic growth of marine ANME consortia was obtained to date. To study possible thermophilic AOM, we investigated hydrothermally influenced sediment from the Guaymas Basin. In vitro incubations showed activity of sulfate-dependent methane oxidation between 5 and 70 °C with an apparent optimum between 45 and 60 °C. AOM was absent at temperatures ⩾75 °C. Long-term enrichment of AOM was fastest at 50 °C, yielding a 13-fold increase of methane-dependent sulfate reduction within 250 days, equivalent to an apparent doubling time of 68 days. The enrichments were dominated by novel ANME-1 consortia, mostly associated with bacterial partners of the deltaproteobacterial HotSeep-1 cluster, a deeply branching phylogenetic group previously found in a butane-amended 60 °C-enrichment culture of Guaymas sediments. The closest relatives (Desulfurella spp.; Hippea maritima) are moderately thermophilic sulfur reducers. Results indicate that AOM and ANME archaea could be of biogeochemical relevance not only in cold to moderate but also in hot marine habitats.     

Genomic markers of ancient anaerobic microbial pathways: sulfate reduction,methanogenesis, and methane oxidation

Genomic markers for anaerobic microbial processes in marine sediments-sulfate reduction, methanogenesis, and anaerobic methane oxidation-reveal the structure of sulfate-reducing, methanogenic, and methane-oxidizing microbial communities (including uncultured members); they allow inferences about the evolution of these ancient microbial pathways; and they open genomic windows into extreme microbial habitats, such as deep subsurface sediments and hydrothermal vents, that are analogs for the early Earth and for extraterrestrial microbiota.     

Assimilation of methane and inorganic carbon by microbial communities mediating the anaerobic oxidation of methane

The anaerobic oxidation of methane (AOM) is a major sink for methane on Earth and is performed by consortia of methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB). Here we present a comparative study using in vitro stable isotope probing to examine methane and carbon dioxide assimilation into microbial biomass. Three sediment types comprising different methane-oxidizing communities (ANME-1 and -2 mixture from the Black Sea, ANME-2a from Hydrate Ridge and ANME-2c from the Gullfaks oil field) were incubated in replicate flow-through systems with methane-enriched anaerobic seawater medium for 5–6 months amended with either ¹³CH₄ or H¹³CO₃^-. In all three sediment types methane was anaerobically oxidized in a 1:1 stoichiometric ratio compared with sulfate reduction. Similar amounts of ¹³CH₄ or ¹³CO₂ were assimilated into characteristic archaeal lipids, indicating a direct assimilation of both carbon sources into ANME biomass. Specific bacterial fatty acids assigned to the partner SRB were almost exclusively labelled by ¹³CO₂, but only in the presence of methane as energy source and not during control incubations without methane. This indicates an autotrophic growth of the ANME-associated SRB and supports previous hypotheses of an electron shuttle between the consortium partners. Carbon assimilation efficiencies of the methanotrophic consortia were low, with only 0.25–1.3 mol% of the methane oxidized.     

Anaerobic methane oxidation and sulfate reduction in bacterial mats of coral-like carbonate structures in the Black Sea

A detailed study of the processes of anaerobic methane oxidation and sulfate reduction in the bacterial mats occurring on coral-like carbonate structures in the region of methane seeps in the Black Sea, as well as of the phenotypic diversity of sulfate-reducing bacteria developing in this zone, has been performed. The use of the radioisotopic method shows the microbial mat structure to be heterogeneous. The peak activity of the two processes was revealed when a mixture of the upper (dark) and underlying (intensely pink) layers was introduced into an incubation flask, which confirms the suggestion that methanotrophic archaea and sulfate-reducing bacteria closely interact in the process of anaerobic methane oxidation. Direct correlation between the rate of anaerobic methane oxidation and the methane and electron acceptor concentrations in the medium has been experimentally demonstrated. Several enrichment and two pure cultures of sulfate-reducing bacteria have been obtained from the near-bottom water and bacterial mats. Both strains were found to completely oxidize the substrates to CO2 and H2S. The bacteria grow at temperatures ranging from -1 to 18 (24) degrees C, with an optimum in the 10-18 degrees C range, and require the presence of 1.5-2.5% NaCl and 0.07-0.2% MgCl2 x 6H2O. Regarding the aggregate of their phenotypic characteristics (cell morphology, spectrum of growth substrates, the capacity for complete oxidation), the microorganisms isolated have no analogues among the psychrophilic sulfate-reducing bacteria already described. The results obtained demonstrate the wide distribution of psychrophilic sulfate-reducing bacteria in the near-bottom water and bacterial mats covering the coral-like carbonate structures occurring in the region of methane seeps in the Black Sea, as well as the considerable catabolic potential of this physiological group of psychrophilic anaerobes in deep-sea habitats.     

海洋氮循环中细菌的厌氧氨氧化

细菌厌氧氨氧化过程是在一类特殊细菌的厌氧氨氧化体内完成的以氨作为电子供体硝酸盐作为电子受体的一种新型脱氮反应.厌氧氨氧化菌的发现,改变人们对传统氮的生物地球化学循环的认识:反硝化细菌并不是大气中氮气产生的唯一生物类群.而且越来越多的证据表明,细菌厌氧氨氧化与全球的氮物质循环密切相关,估计海洋细菌的厌氧氨氧化过程占到全球海洋氮气产生的一半左右.由于氮与碳的循环密切相关,因此可以推测,细菌的厌氧氨氧化会影响大气中的二氧化碳浓度,从而对全球气候变化产生重要影响.另外,由于细菌厌氧氨氧化菌实现了氨氮的短程转化,缩短了氮素的转化过程,因此为开发更节约能源、更符合可持续发展要求的废水脱氮新技术提供了生物学基础.     

An inorganic geochemical argument for coupled anaerobic oxidation of methane and iron reduction in marine sediments

Here, we present results from sediments collected in the Argentine Basin, a non‐steady state depositional marine system characterized by abundant oxidized iron within methane‐rich layers due to sediment reworking followed by rapid deposition. Our comprehensive inorganic data set shows that iron reduction in these sulfate and sulfide‐depleted sediments is best explained by a microbially mediated process—implicating anaerobic oxidation of methane coupled to iron reduction (Fe‐AOM) as the most likely major mechanism. Although important in many modern marine environments, iron‐driven AOM may not consume similar amounts of methane compared with sulfate‐dependent AOM. Nevertheless, it may have broad impact on the deep biosphere and dominate both iron and methane cycling in sulfate‐lean marine settings. Fe‐AOM might have been particularly relevant in the Archean ocean, >2.5 billion years ago, known for its production and accumulation of iron oxides (in iron formations) in a biosphere likely replete with methane but low in sulfate. Methane at that time was a critical greenhouse gas capable of sustaining a habitable climate under relatively low solar luminosity, and relationships to iron cycling may have impacted if not dominated methane loss from the biosphere.     

Effect of methanogenic substrates on anaerobic oxidation of methane and sulfate reduction by an anaerobic methanotrophic enrichment

Anaerobic oxidation of methane (AOM) coupled to sulfate reduction (SR) is assumed to be a syntrophic process, in which methanotrophic archaea produce an interspecies electron carrier (IEC), which is subsequently utilized by sulfate-reducing bacteria. In this paper, six methanogenic substrates are tested as candidate-IECs by assessing their effect on AOM and SR by an anaerobic methanotrophic enrichment. The presence of acetate, formate or hydrogen enhanced SR, but did not inhibit AOM, nor did these substrates trigger methanogenesis. Carbon monoxide also enhanced SR but slightly inhibited AOM. Methanol did not enhance SR nor did it inhibit AOM, and methanethiol inhibited both SR and AOM completely. Subsequently, it was calculated at which candidate-IEC concentrations no more Gibbs free energy can be conserved from their production from methane at the applied conditions. These concentrations were at least 1,000 times lower can the final candidate-IEC concentration in the bulk liquid. Therefore, the tested candidate-IECs could not have been produced from methane during the incubations. Hence, acetate, formate, methanol, carbon monoxide, and hydrogen can be excluded as sole IEC in AOM coupled to SR. Methanethiol did inhibit AOM and can therefore not be excluded as IEC by this study.     

In vitro cell growth of marine archaeal-bacterial consortia during anaerobic oxidation of methane with sulfate

Anoxic sediment from a methane hydrate area (Hydrate Ridge, north-east Pacific; water depth 780 m) was incubated in a long-term laboratory experiment with semi-continuous supply of pressurized [1.4 MPa (14 atm)] methane and sulfate to attempt in vitro propagation of the indigenous consortia of archaea (ANME-2) and bacteria (DSS, Desulfosarcina/Desulfococcus cluster) to which anaerobic oxidation of methane (AOM) with sulfate has been attributed. During 24 months of incubation, the rate of AOM (measured as methane-dependent sulfide formation) increased from 20 to 230 micromol day(-1) (g sediment dry weight)(-1) and the number of aggregates (determined by microscopic counts) from 0.5 x 10(8) to 5.7 x 10(8) (g sediment dry weight)(-1). Fluorescence in situ hybridization targeting 16S rRNA of both partners showed that the newly grown consortia contained central archaeal clusters and peripheral bacterial layers, both with the same morphology and phylogenetic affiliation as in the original sediment. The development of the AOM rate and the total consortia biovolume over time indicated that the consortia grew with a doubling time of approximately 7 months (growth rate 0.003 day(-1)) under the given conditions. The molar growth yield of AOM was approximately 0.6 g cell dry weight (mol CH(4) oxidized)(-1); according to this, only 1% of the consumed methane is channelled into synthesis of consortia biomass. Concentrations of biomarker lipids previously attributed to ANME-2 archaea (e.g. sn-2-hydroxyarchaeol, archaeol, crocetane, pentamethylicosatriene) and Desulfosarcina-like bacteria [e.g. hexadecenoic-11 acid (16:1omega5c), 11,12-methylene-hexadecanoic acid (cy17:0omega5,6)] strongly increased over time (some of them over-proportionally to consortia biovolume), suggesting that they are useful biomarkers to detect active anaerobic methanotrophic consortia in sediments.     

Diel interactions of oxygenic photosynthesis and n(2) fixation (acetylene reduction) in a marine microbial mat community

Diel variations in N(2) fixation (acetylene reduction), CO(2) fixation, and oxygen concentrations were measured, on three separate occasions, in a marine microbial mat located on Shackleford Banks, North Carolina. Nitrogenase activity (NA) was found to be inversely correlated with CO(2) fixation and, in two of the three diel periods studied, was higher at night than during the day. Oxygen concentrations within the top 3 mm of the mat ranged from 0 to 400 muM on a diel cycle; anaerobic conditions generally persisted below 4 mm. NA in the mat was profoundly affected by naturally occurring oxygen concentrations. Experimentally elevated oxygen concentrations resulted in a significant depression of NA, whereas the addition of the Photosystem II inhibitor 3(3,4-dichlorophenyl)-1,1-dimethylurea decreased oxygen concentrations within the mat and resulted in a significant short-term enhancement of NA. Mat N(2)-fixing microorganisms include cyanobacteria and heterotrophic, photoautotrophic, and chemolithotrophic eubacteria. Measured (whole-mat) NA is probably due to a combination of the NA of each of these groups of organisms. The relative contributions of each group to whole-mat NA probably varied during diel and seasonal (successional) cycles. Reduced compounds derived from photosynthetic CO(2) fixation appeared to be an important source of energy for NA during the day, whereas heterotrophic or chemolithotrophic utilization of reduced compounds appeared to be an important source of energy for NA at night, under reduced ambient oxygen concentrations. Previous estimates of N(2) fixation calculated on the basis of daytime measurements may have seriously underestimated diel and seasonal nitrogen inputs in mat systems.     

Biogeochemical and molecular signatures of anaerobic methane oxidation in a marine sediment

Anaerobic methane oxidation was investigated in 6-m-long cores of marine sediment from Aarhus Bay, Denmark. Measured concentration profiles for methane and sulfate, as well as in situ rates determined with isotope tracers, indicated that there was a narrow zone of anaerobic methane oxidation about 150 cm below the sediment surface. Methane could account for 52% of the electron donor requirement for the peak sulfate reduction rate detected in the sulfate-methane transition zone. Molecular signatures of organisms present in the transition zone were detected by using selective PCR primers for sulfate-reducing bacteria and for Archaea. One primer pair amplified the dissimilatory sulfite reductase (DSR) gene of sulfate-reducing bacteria, whereas another primer (ANME) was designed to amplify archaeal sequences found in a recent study of sediments from the Eel River Basin, as these bacteria have been suggested to be anaerobic methane oxidizers (K. U. Hinrichs, J. M. Hayes, S. P. Sylva, P. G. Brewer, and E. F. DeLong, Nature 398:802-805, 1999). Amplification with the primer pairs produced more amplificate of both target genes with samples from the sulfate-methane transition zone than with samples from the surrounding sediment. Phylogenetic analysis of the DSR gene sequences retrieved from the transition zone revealed that they all belonged to a novel deeply branching lineage of diverse DSR gene sequences not related to any previously described DSR gene sequence. In contrast, DSR gene sequences found in the top sediment were related to environmental sequences from other estuarine sediments and to sequences of members of the genera Desulfonema, Desulfococcus, and Desulfosarcina. Phylogenetic analysis of 16S rRNA sequences obtained with the primers targeting the archaeal group of possible anaerobic methane oxidizers revealed two clusters of ANME sequences, both of which were affiliated with sequences from the Eel River Basin.     

Diversity and abundance of sulfate-reducing microorganisms in the sulfate and methane zones of a marine sediment, Black Sea

The Black Sea, with its highly sulfidic water column, is the largest anoxic basin in the world. Within its sediments, the mineralization of organic matter occurs essentially through sulfate reduction and methanogenesis. In this study, the sulfate-reducing community was investigated in order to understand how these microorganisms are distributed relative to the chemical zonation: in the upper sulfate zone, at the sulfate-methane transition zone, and deeply within the methane zone. Total bacteria were quantified by real-time PCR of 16S rRNA genes whereas sulfate-reducing microorganisms (SRM) were quantified by targeting their metabolic key gene, the dissimilatory (bi)sulfite reductase (dsrA). Sulfate-reducing microorganisms were predominant in the sulfate zone but occurred also in the methane zone, relative proportion was maximal around the sulfate-methane transition, c. 30%, and equally high in the sulfate and methane zones, 5-10%. The dsrAB clone library from the sulfate-methane transition zone, showed mostly sequences affiliated with the Desulfobacteraceae. While, the dsrAB clone libraries from the upper, sulfate-rich zone and the deep, sulfate-poor zone were dominated by similar, novel deeply branching sequences which might represent Gram-positive spore-forming sulfate- and/or sulfite-reducing microorganisms. We thus hypothesize that terminal carbon mineralization in surface sediments of the Black Sea is largely due to the sulfate reduction activity of previously hidden SRM. Although these novel SRM were also abundant in sulfate-poor, methanogenic areas of the Black Sea sediment, their activities and possibly very versatile metabolic capabilities remain subject of further study.     

In vitro demonstration of anaerobic oxidation of methane coupled to sulphate reduction in sediment from a marine gas hydrate area

Anaerobic oxidation of methane (AOM) and sulphate reduction were examined in sediment samples from a marine gas hydrate area (Hydrate Ridge, NE Pacific). The sediment contained high numbers of microbial consortia consisting of organisms that affiliate with methanogenic archaea and with sulphate-reducing bacteria. Sediment samples incubated under strictly anoxic conditions in defined mineral medium (salinity as in seawater) produced sulphide from sulphate if methane was added as the sole organic substrate. No sulphide production occurred in control experiments without methane. Methane-dependent sulphide production was fastest between 4 degree C and 16 degree C, the average rate with 0.1 MPa (approximately 1 atm) methane being 2.5 micro mol sulphide day(-1) and (g dry mass sediment)(-1). An increase of the methane pressure to 1.1 MPa (approximately 11 atm) resulted in a four to fivefold increase of the sulphide production rate. Quantitative measurements using a special anoxic incubation device without gas phase revealed continuous consumption of dissolved methane (from initially 3.2 to 0.7 mM) with simultaneous production of sulphide at a molar ratio of nearly 1:1. To test the response of the indigenous community to possible intermediates of AOM, molecular hydrogen, formate, acetate or methanol were added in the absence of methane; however, sulphide production from sulphate with any of these compounds was much slower than with methane. In the presence of methane, such additions neither stimulated nor inhibited sulphate reduction. Hence, the experiments did not provide evidence for one of these compounds acting as a free extracellular intermediate (intercellular shuttle) during AOM by the presently investigated consortia.     

Intensities of microbial production and oxidation of methane in bottom sediments and water mass of the Black Sea

Intensities of biogeochemical (microbial) processes of methane production and methane oxidation were determined in bottom sediments and water column of the Black Sea. Aerobic bacterial oxidation of methane is confined to the upper 20-30 cm of Holocene bottom sediments of the shelf (0.7-259 ng C/(dm3 day)) and oxygenated waters (0.2-45 ng C/(dm3 day)). In reduced sediments of the deep-sea zone and in the hydrogen sulfide-containing water column, considerable intensities of anaerobic methane oxidation were recorded, comparable to or exceeding the intensities of methane oxidation in oxygenated layers. From one fourth to one half of the methane formed in bottom sediments was oxidized immediately therein. The major part of the remaining methane was oxidized in the water column, and a smaller portion arrived in the atmosphere.     

微生物介导的甲烷厌氧氧化过程及其影响因子研究进展

温室气体甲烷减排是全球变化领域的研究热点,甲烷厌氧氧化(anaerobic methane oxidation,AOM)过程是一个以前被忽视的甲烷汇,在调控全球甲烷收支平衡及减缓温室效应等方面扮演着十分重要的角色。AOM微生物以甲烷为唯一电子供体,与硫酸盐(SO42-)、亚硝酸盐(NO2-)/硝酸盐(NO3-)、金属离子(Fe3+、Mn4+、Cr6+)等结合完成氧化还原过程,该过程是耦合碳、氮、硫循环的关键环节。本文系统整理分析了不同AOM类型、发生机理、相关功能微生物类群(ANME-1、ANME-2、ANME-3、NC10、MBG-D)及影响AOM过程的关键调控因子的最新研究进展。结果发现,目前80%以上研究都集中在对最常见电子受体类型(SO42-/NO3-/NO2-/Fe3+/Mn4+)的AOM相关过程,而忽视了潜在的新型电子受体(AQDS/HAs O42-/Cr6+/ClO4-等)的耦合作用过程和相对应的微生物类型及作用机理。对未来AOM研究方向提出展望,以期为研究甲烷厌氧氧化菌在不同生态系统中的生态分布及减缓全球温室气体排放提供新的思路。     

Effects of temperature and pressure on sulfate reduction and anaerobic oxidation of methane in hydrothermal sediments of Guaymas Basin

Rates of sulfate reduction (SR) and anaerobic oxidation of methane (AOM) in hydrothermal deep-sea sediments from Guaymas Basin were measured at temperatures of 5 to 200 degrees C and pressures of 1 x 10(5), 2.2 x 10(7), and 4.5 x 10(7) Pa. A maximum SR of several micromoles per cubic centimeter per day was found at between 60 and 95 degrees C and 2.2 x 10(7) and 4.5 x 10(7) Pa. Maximal AOM was observed at 35 to 90 degrees C but generally accounted for less than 5% of SR.     

A thermodynamic analysis of the anaerobic oxidation of methane in marine sediments

Anaerobic oxidation of methane (AOM) in anoxic marine sediments is a significant process in the global methane cycle, yet little is known about the role of bulk composition, temperature and pressure on the overall energetics of this process. To better understand the biogeochemistry of AOM, we have calculated and compared the energetics of a number of candidate reactions that microorganisms catalyse during the anaerobic oxidation of methane in (i) a coastal lagoon (Cape Lookout Bight, USA), (ii) the deep Black Sea, and (iii) a deep-sea hydrothermal system (Guaymas basin, Gulf of California). Depending on the metabolic pathway and the environment considered, the amount of energy available to the microorganisms varies from 0 to 184 kJ mol(-1). At each site, the reactions in which methane is either oxidized to HCO3(-), acetate or formate are generally only favoured under a narrow range of pressure, temperature and solution composition--particularly under low (10(-10 )m) hydrogen concentrations. In contrast, the reactions involving sulfate reduction with H2, formate and acetate as electron donors are nearly always thermodynamically favoured. Furthermore, the energetics of ATP synthesis was quantified per mole of methane oxidized. Depending on depth, between 0.4 and 0.6 mol of ATP (mol CH4(-1) was produced in the Black Sea sediments. The largest potential productivity of 0.7 mol of ATP (mol CH4(-1) was calculated for Guaymas Basin, while the lowest values were predicted at Cape Lookout Bight. The approach used in this study leads to a better understanding of the environmental controls on the energetics of AOM.     

Production of N(2) through anaerobic ammonium oxidation coupled to nitrate reduction in marine sediments

In the global nitrogen cycle, bacterial denitrification is recognized as the only quantitatively important process that converts fixed nitrogen to atmospheric nitrogen gas, N(2), thereby influencing many aspects of ecosystem function and global biogeochemistry. However, we have found that a process novel to the marine nitrogen cycle, anaerobic oxidation of ammonium coupled to nitrate reduction, contributes substantially to N(2) production in marine sediments. Incubations with (15)N-labeled nitrate or ammonium demonstrated that during this process, N(2) is formed through one-to-one pairing of nitrogen from nitrate and ammonium, which clearly separates the process from denitrification. Nitrite, which accumulated transiently, was likely the oxidant for ammonium, and the process is thus similar to the anammox process known from wastewater bioreactors. Anaerobic ammonium oxidation accounted for 24 and 67% of the total N(2) production at two typical continental shelf sites, whereas it was detectable but insignificant relative to denitrification in a eutrophic coastal bay. However, rates of anaerobic ammonium oxidation were higher in the coastal sediment than at the deepest site and the variability in the relative contribution to N(2) production between sites was related to large differences in rates of denitrification. Thus, the relative importance of anaerobic ammonium oxidation and denitrification in N(2) production appears to be regulated by the availability of their reduced substrates. By shunting nitrogen directly from ammonium to N(2), anaerobic ammonium oxidation promotes the removal of fixed nitrogen in the oceans. The process can explain ammonium deficiencies in anoxic waters and sediments, and it may contribute significantly to oceanic nitrogen budgets.     

Influence of sulfate reduction on the microbial dechlorination of pentachloroaniline in a mixed anaerobic culture

The reductive dechlorination of pentachloroaniline (PCA) was investigated in the absence and presence of sulfate in batch assays using a PCA-dechlorinating mixed anaerobic culture with methanol as the external electron donor at neutral pH and 22°C. PCA at an initial concentration of 7.8 μM was sequentially dechlorinated to dichlorinated anilines in the sulfate-free culture and the culture amended with 300 mg sulfate-S/L. At an initial concentration of 890 mg sulfate-S/L, a higher sulfate reduction rate was achieved, but PCA dechlorination was not observed until the sulfate concentration dropped to about 100 mg S/L. The transient inhibition of PCA is attributed to competition between sulfate reducing and dechlorinating species for electron donor, more likely for H₂ resulting from methanol fermentation. A long-term (118 days) PCA dechlorination assay with the sulfate-amended culture, which included five feeding cycles, resulted in accumulation of both sulfide (886 mg S/L) and acetate (1,900 mg COD/L). Under these conditions, the sulfate reducers were inhibited, while the rate and pathway of PCA dechlorination were not affected. The results of this study show that the rate of sulfate reduction rather than the sulfate concentration alone dictates the outcome of the competition between sulfate reducers and either dechlorinators or methanogens. The findings of the present study have significant implications relative to the fate and transport of PCA and its dechlorination products in sulfate-laden subsurface systems.